The pssB gene product of Rhizobium leguminosarum bv. trifolii is homologous to a family of inositol monophosphatases

The Rhizobium leguminosarum bv. trifolii region encoding pssA and pssB genes was cloned. The pssB gene located upstream of the pssA encoded a 28.36-kDa protein which displayed 97.5% identity with the PssB of R. leguminosarum bv. viciae. Inactivation of the pssB gene by insertion of the lacZ-Gmr cassette resulted in the significant increased production of exopolysaccharide in comparison to the wild-type level. A mutant strain was also defective in nitrogen fixation suggesting a regulatory role of pssB in symbiosis with clover.     

PssO, a unique extracellular protein important for exopolysaccharide synthesis in Rhizobium leguminosarum bv. trifolii

Synthesis and secretion of polysaccharides by Gram-negative bacteria are a result of a concerted action of enzymatic and channel-forming proteins localized in different compartments of the cell. The presented work comprises functional characterization of PssO protein encoded within the previously identified, chromosomal exopolysaccharide (EPS) biosynthesis region (Pss-I) of symbiotic bacterium Rhizobium leguminosarum bv. trifolii TA1 (RtTA1). pssO gene localization between pssN and pssP genes encoding proteins engaged in exopolysaccharide synthesis and transport, suggested its role in EPS synthesis and/or secretion. RtTA1 pssO deletion mutant and the PssO protein overproducing strains were constructed. The mutant strain was EPS-deficient, however, this mutation was not complemented. The PssO-overproducing strain was characterized by increase in EPS secretion. Subcellular fractionation, pssO-phoA/lacZ translational fusion analyses and immunolocalisation of PssO on RtTA1 cell surface by electron microscopy demonstrated that PssO is secreted to the extracellular medium and remains attached to the cell. Western blotting analysis revealed the presence of immunologically related proteins within the species R. leguminosarum bv. trifolii, bv. viciae and Rhizobium etli. The secondary structure of PssO-His(6), as determined by FTIR spectroscopy, consists of at least 32% alpha-helical and 12% beta-sheet structures. A putative function of PssO in EPS synthesis and/or transport is discussed in the context of its cellular localization and the phenotypes of the deletion mutant and pssO-overexpressing strain.     

Distribution of O-acetyl groups in the exopolysaccharide synthesized by Rhizobium leguminosarum strains is not determined by the Sym plasmid

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.     

A cultivar-specific interaction between Rhizobium leguminosarum bv. trifolii and subterranean clover is controlled by nodM, other bacterial cultivar specificity genes, and a single recessive host gene.

Insertion mutagenesis identified two negatively acting gene loci which restrict the ability of Rhizobium leguminosarum bv. trifolii TA1 to infect the homologous host Trifolium subterraneum cv. Woogenellup. One locus was confirmed by DNA sequence analysis as the nodM gene, while the other locus, designated csn-1 (cultivar-specific nodulation), is not located on the symbiosis plasmid. The presence of these cultivar specificity loci could be suppressed by the introduction of the nodT gene from ANU843, a related R. leguminosarum bv. trifolii strain. Other nod genes, present in R. leguminosarum bv. viciae (including nodX) and R. meliloti, were capable of complementing R. leguminosarum bv. trifolii TA1 for nodulation on cultivar Woogenellup. Nodulation studies conducted with F2 seedlings from a cross between cultivar Geraldton and cultivar Woogenellup indicated that a single recessive gene, designated rwt1, is responsible for the Nod- association between strain TA1 and cultivar Woogenellup. Parallels can be drawn between this association and gene-for-gene systems common in interactions between plants and biotrophic pathogens.     

Rhizobium leguminosarum bv. trifolii PssP protein is required for exopolysaccharide biosynthesis and polymerization

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) that is important for the induction of nitrogen-fixing nodules on clover. Recently, three genes, pssN, pssO, and pssP, possibly involved in EPS biosynthesis and polymerization were identified. The predicted protein product of the pssP gene shows a significant sequence similarity to other proteins belonging to the PCP2a family that are involved in the synthesis of high-molecular-weight EPS. An R. leguminosarum bv. trifolii TA1 mutant with the entire coding region of pssP deleted did not produce the EPS. A pssP mutant with the 5' end of the gene disrupted produced exclusively low-molecular-weight EPS. A mutant that synthesized a functional N-terminal periplasmic domain but lacked the C-terminal part of PssP produced significantly reduced amounts of EPS with a slightly changed low to high molecular form ratio. Mutants affected in the PssP protein carrying a stable plasmid with a constitutively expressed gusA gene induced nodules on red clover that were not fully occupied by bacteria. A mutant with the entire pssP gene deleted infected only a few plant cells in the nodule. The pssP promoter-gusA reporter fusion was active in bacteroids during nodule development.     

Exopolysaccharides of Rhizobium leguminosarum biovar trifolii harbouring cloned exo region.

Rhizobium leguminosarum bv. trifolii produces an acidic exopolysaccharide (EPS) which plays an important role in the development of nitrogen-fixing nodules. Tn5 mutant of R. trifolii 93 defective in EPS production (Exo-) forms ineffective (Fix-) nodules on red clover. This Exo- mutation is complemented by the pARF1368 and pARF25 cosmids isolated from gene bank of Rhizobium trifolii TA1, but the complementation is not correlated with restoration of Fix+ phenotype. Furthermore, these cosmids introduced to wild-type of R. trifolii 24 repress its ability to form nitrogen-fixing nodules. These results might suggest that bacteria with cosmids carrying the exo region form EPS of altered structure. It has been shown by 1H-n.m.r. that exopolysaccharides produced by R. trifolii 93pARF-1368 and 93pARF25 contain less non-carbohydrate residues (acetyl, pyruvyl and 3-hydroxybutanoyl) than the wild type EPS. These data suggest that the biological activity of the exopolysaccharide of R. trifolii depends on the contents of the non-carbohydrate substitutions.     

Elevated levels of synthesis of over 20 proteins results after mutation of the Rhizobium leguminosarum exopolysaccharide synthesis gene pssA

The protein expression profiles of Rhizobium leguminosarum strains in response to specific genetic perturbations in exopolysaccharide (EPS) biosynthesis genes were examined using two-dimensional gel electrophoresis. Lesions in either pssA, pssD, or pssE of R. leguminosarum bv. viciae VF39 or in pssA of R. leguminosarum bv. trifolii ANU794 not only abolished the capacity of these strains to synthesize EPS but also had a pleiotropic effect on protein synthesis levels. A minimum of 22 protein differences were observed for the two pssA mutant strains. The differences identified in the pssD and pssE mutants of strain VF39 were a distinct subset of the same protein synthesis changes that occurred in the pssA mutant. The pssD and pssE mutant strains shared identical alterations in the proteins synthesized, suggesting that they share a common function in the biosynthesis of EPS. In contrast, a pssC mutant that produces 38% of the EPS level of the parental strain showed no differences in its protein synthesis patterns, suggesting that the absence of EPS itself was contributing to the changes in protein synthesis and that there may be a complex interconnection of the EPS biosynthetic pathway with other metabolic pathways. Genetic complementation of pssA can restore wild-type protein synthesis levels, indicating that many of the observed differences in protein synthesis are also a specific response to a dysfunctional PssA. The relevance of these proteins, which are grouped as members of the pssA mutant stimulon, remains unclear, as the majority lacked a homologue in the current sequence databases and therefore possibly represent a novel functional network(s). These findings have illustrated the potential of proteomics to reveal unexpected higher-order processes of protein function and regulation that arise from mutation. In addition, it is evident that enzymatic pathways and regulatory networks are more interconnected and more sensitive to structural changes in the cell than is often appreciated. In these cases, linking the observed phenotype directly to the mutated gene can be misleading, as the phenotype could be attributable to downstream effects of the mutation.     

Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: subcellular localization and possible involvement in exopolysaccharide export

Surface expression of exopolysaccharides (EPS) in gram-negative bacteria depends on the activity of proteins found in the cytoplasmic membrane, the periplasmic space, and the outer membrane. pssTNOP genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might be components of the EPS polymerization and secretion system. In this study, we have characterized PssN protein. Employing pssN-phoA and pssN-lacZ gene fusions and in vivo acylation with [3H]palmitate, we demonstrated that PssN is a 43-kDa lipoprotein directed to the periplasm by an N-terminal signal sequence. Membrane detergent fractionation followed by sucrose gradient centrifugation showed that PssN is an outer membrane-associated protein. Indirect immunofluorescence with anti-PssN and fluorescein isothiocyanate-conjugated antibodies and protease digestion of spheroplasts and intact cells of TA1 provided evidence that PssN is oriented towards the periplasmic space. Chemical cross-linking of TA1 and E. coli cells overproducing PssN-His6 protein showed that PssN might exist as a homo-oligomer of at least two monomers. Investigation of the secondary structure of purified PssN-His6 protein by Fourier transform infrared spectroscopy revealed the predominant presence of beta-structure; however, alpha-helices were also detected. Influence of an increased amount of PssN protein on the TA1 phenotype was assessed and correlated with a moderate enhancement of EPS production.     

Membrane topology of PssT,the transmembrane protein component of the type I exopolysaccharide transport system in Rhizobium leguminosarum bv. trifolii strain TA1

The pssT gene was identified as the fourth gene located upstream of the pssNOP gene cluster possibly involved in the biosynthesis, polymerization, and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum bv. trifolii strain TA1. The hydropathy profile and homology searches indicated that PssT belongs to the polysaccharide-specific transport family of proteins, a component of the type I system of the polysaccharide transport. The predicted membrane topology of the PssT protein was examined with a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions. The results generally support a predicted topological model for PssT consisting of 12 transmembrane segments, with amino and carboxyl termini located in the cytoplasm. A mutant lacking the C-terminal part of PssT produced increased amounts of total EPS with an altered distribution of high- and low-molecular-weight forms in comparison to the wild-type RtTA1 strain. The PssT mutant produced an increased number of nitrogen fixing nodules on clover.     

The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N-terminal domain found in other NifA proteins

The nifA gene has been identified between the fixX and nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii (R.I. bv. trifolii) strain ANU843. Expression of the nifA gene is induced in the symbiotic state and site-directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation. Interestingly, the predicted R.I. bv. trifolii NifA protein lacks an N-terminal domain that is present in the homologous proteins from R.I. bv. viciae, Rhizobium meliloti, Bradyrhizobium japonicum, Klebsiella pneumoniae and all other documented NifA proteins. This indicates that this N-terminal domain is not essential for NifA function in R.I. bv. trifolii.     

Cloning and characterization of ce/8A gene from Rhizobium leguminosarum bv. trifolii 1536

AIMS: To isolate the cellulase gene from Rhizobium leguminosarum bv. trifolii 1536. METHODS AND RESULTS: By the shot-gun method a clone (cel8A) harbouring 3.1 kb genomic DNA fragment from R. leguminosarum bv. trifolii 1536 was obtained. The cel8A gene coded 348 amino acids and it belongs to the glycosyl hydrolase family 8. The molecular mass of Cel8A protein induced from Escherichia coli DH5alpha, appeared to be 35 kDa. The optimum pH and optimum temperature was 7.0, and about 30 degrees C for its enzymatic activity respectively. CONCLUSIONS: R. leguminosarum bv. trifolii 1536 had cel8A gene having an open reading frame of 1047 bp coded for the activity of hydrolyzation of carboxymethyl cellulose. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of celluloytic enzyme by R. leguminosarum bv. trifolii was confirmed, which would play specific roles in rhizobia. Future study should focus on its role in the infection and nodulation phenomena.     

Host-range related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum

Proton nuclear magnetic resonance (1H NMR) and fast atom bombardment mass spectrometric analyses were performed on enzymatically derived oligosaccharides from the acidic excreted polysaccharides (EPS) from representative bacterial strains of the pea-nodulating symbiont, Rhizobium leguminosarum (128C53, 128C63, and 300) and the clover-nodulating symbiont, Rhizobium trifolii (NA-30, ANU843, 0403, TA-1, LPR5035, USDA20.102, and 4S). The results revealed structural similarities and differences between EPS of these two species. Octasaccharide units containing galactose, glucuronic acid, alpha-L-threo-hex-4-enopyranosyluronic acid, and glucose in a molar ratio of 1:1:1:5 were obtained from the EPS of the three R. leguminosarum strains and had the same primary glycosyl sequence and location of pyruvate, acetate, and 3-hydroxybutyrate substituents. About 80% of the galactose residues were acylated with 3-hydroxybutyrate, and there were two acetyl groups per repeating unit distributed between the 2 glucose residues of the main chain-derived sequence of the octasaccharides. In contrast, the R. trifolii strains had varied EPS structures, each of which differed from the common R. leguminosarum EPS structure. The EPS from one group of R. trifolii strains (0403 and LPR5035) most closely resembled the R. leguminosarum EPS but differed in that a lower number of galactose and glucose residues were substituted by 3-hydroxybutyryl and acetyl groups, respectively. The EPS from a second group of R. trifolii strains (ANU843, TA-1, and NA-30) was even more different than the R. leguminosarum EPS. These R. trifolii octasaccharides bore a single acetyl group on O-3 of the glucuronic acid residue. In addition, the level of acylation by 3-hydroxybutyryl groups was 50% of that present in the R. leguminosarum EPS. The remaining two strains of R. trifolii (USDA20.102 and 4S) had very different patterns of acylation to each other and to all of the other strains. The EPS from strain USDA20.102 practically lacked 3-hydroxybutyryl groups and had a unique degree and pattern of acetylation. The oligomers from the EPS of R. trifolii strain 4S completely lacked 3-hydroxybutyryl groups and galactose. The latter EPS contained only one O-1-carboxyethylidene group and had a different degree and pattern of acetylation. Interestingly, these two latter strains differ from the other R. trifolii strains in nodulation rates on rare clover species in the Trifolium cross-inoculation group. Thus, we define several groups of R. trifolii based upon their EPS structures and establish their similarities and distinct differences with the EPS of R. leguminosarum.(ABSTRACT TRUNCATED AT 400 WORDS)     

The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation

The effect of the rhizobium adhesion protein RapA1 on Rhizobium leguminosarum bv. trifolii adsorption to Trifolium pratense (red clover) roots was investigated. We altered RapA1 production by cloning its encoding gene under the plac promoter into the stable vector pHC60. After introducing this plasmid in R. leguminosarum bv. trifolii, three to four times more RapA1 was produced, and two to five times higher adsorption to red clover roots was obtained, as compared with results for the empty vector. Enhanced adsorption was also observed on soybean and alfalfa roots, not related to R. leguminosarum cross inoculation groups. Although the presence of 1 mM Ca2+ during rhizobial growth enhanced adsorption, it was unrelated to RapA1 level. Similar effects were obtained when the same plasmid was introduced in Rhizobium etli for its adsorption to bean roots. Although root colonization by the RapA1-overproducing strain was also higher, nodulation was not enhanced. In addition, in vitro biofilm formation was similar to the wild-type both on polar and on hydrophobic surfaces. These results suggest that RapA1 receptors are present in root but not on inert surfaces, and that the function of this protein is related to rhizosphere colonization.     

Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv. trifolii R39 studied with monospecific polyclonal antisera.

Monospecific polyclonal antisera raised against Rhizobium leguminosarum bv. trifolii R39, a bacterium which was isolated originally from red clover nodules, were used to study the colonization of roots of leguminous and nonleguminous plants (Pisum sativum, Lupinus albus, Triticúm aestivum, and Zea mays) after inoculation. Eight weeks after inoculation of soil-grown plants, between 0.1 and 1% of the total bacterial population in the rhizospheres of all inoculated plants were identified as R. leguminosarum bv. trifolii R39. To characterize the associative colonization of the nonleguminous plants by R.leguminosarum bv. trifolii R39 in more detail, a time course study was performed with inoculated roots of Z. mays. R. leguminosarum bv. trifolii R39 was found almost exclusively in the rhizosphere soil and on the rhizoplane 4 weeks after inoculation. Colonization of inner root tissues was detected only occasionally at this time. During the process of attachment of R. leguminosarum bv. trifolii R39 to the rhizoplane, bacterial lipopolysaccharides were overexpressed, and this may be important for plant-microbe interaction. Fourteen weeks after inoculation, microcolonies of R. leguminosarum bv. trifolii R39 were detected in lysed cells of the root cortex as well as in intracellular space of central root cylinder cells. At the beginning of flowering (18 weeks after inoculation), the number of R. leguminosarum bv. trifolii R39 organisms decreased in the rhizosphere soil, rhizoplane, and inner root tissue.     

Topological and transcriptional analysis of <Emphasis Type="Italic">pssL</Emphasis> gene product: a putative Wzx-like exopolysaccharide translocase in <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii</Emphasis> TA1

An identified pssL gene is yet another one, besides the pssT, pssN and pssP genes, encoding for a protein engaged in polysaccharide polymerization and export in Rhizobium leguminosarum bv. trifolii strain TA1 (RtTA1). Amino acid sequence similarity and hypothetical protein secondary structure placed the PssL protein within Wzx (RfbX) translocases with putative flippase function that belong to the polysaccharide specific transport (PST) family. The predicted secondary structure of the PssL membrane protein was examined with a series of PssL-PhoA and PssL-LacZ translational fusions. The results support the hypothesis of PssL being a member of PST protein family comprising transporters with 12 membrane spanning segments and amino and carboxyl termini located in the cytoplasm. Results of semi-quantitative RT-PCR showed that the initial abundance of mRNA encoding PssL protein was relatively lower when compared to the quantity of the previously identified PssT membrane protein. PssL might be a good candidate for Wzx-like protein that together with PssT (Wzy protein) could be responsible for Wzx/Wzy-like-dependent EPS polymerization and translocation in RtTA1.